结核分枝杆菌H37有毒株与无毒株的代谢相关基因pabB和lpdA启动子活性分析

Comparison of the promoter activity of metabolism-related pabB and lpdA genes between Mycobacterium tuberculosis strains H37Rv and H37Ra

【目的】通过分析结核分枝杆菌无毒株H37Ra的全基因组序列,并与H37Rv基因组序列比较,发现pabB和lpdA预测的启动子区发生了突变。我们利用报告基因,确认启动子突变与其基因转录水平的关系,探索结核分枝杆菌H37Ra毒力丧失的内在原因。【方法】利用生物信息学方法预测这两对基因的启动子区,采用PCR技术克隆这两对基因的启动子,与分枝杆菌启动子探针载体pMC210相连,DNA测序证实连接片段正确后,用电穿孔法将重组质粒转化至耻垢分枝杆菌mc2155。利用Quantitative Real-Time RT-PCR检测报告基因lacZ转录水平的差异,进一步验证这两对基因启动子的突变对相应基因转录水平的影响。【结果】Quantitative Real Time PCR检测结果显示H37RapabB启动子活性是H37Rv pabB启动子活性的6倍(p<0.05),而H37Rv lpdA启动子的活性是H37RalpdA启动子的2倍(p<0.05)。【结论】pabB,lpdA的启动子在H37Ra中的突变对其启动子的活性产生了影响,其中lpdA启动子的突变可能与结核分枝杆菌H37Ra的毒力丧失有关。

Objective Comparative genomic study revealed mutations at the promoter regions of pabB and lpdA in avirulent Mycobacterium tuberculosis strain H37Ra compared to its virulent counterpart strain H37Rv.We used LacZ reporter system to test whether those mutations will affect promoter activity and its potential relationship to virulence attenuation of H37Ra.MethodsPromoter regions of pabB and lpdA were predicted by the＂Neural Network Promoter Prediction＂method（http：/ /www.fruitfly.org /seq_tools /promoter.html）.Promoter sequences were PCR amplified and cloned into mycobacterial promoterless probe vector pMC210.Resultant recombinant plasmids were transformed into M.smegmatis by electroporation.The transcription activity of lacZ under the control of cloned promoters were monitored by Quantitative Real-Time RT-PCR.ResultsQuantitative Real Time PCR results showed that the promoter activity of H37Ra pabB was six times more than that of H37Rv pabB（p 0.05）,while the promoter activity of the H37Rv lpdA was two fold of that of H37Ra lpdA（p 0.05 ）.Conclusion The mutations in pabB and lpdA promoters affect its expression activity.The T-A mutation in lpdA promoter may be related to virulence attenuation of H37Ra.